Thioesterases I and II terminate fatty acid chain growth during fatty acid biosynthesis by hydrolysis of the acyl thioester linkage between the fatty acid and the phosphopantetheine cofactor of fatty acid synthase. A crystallographic study of thioesterase II was initiated in order to better understand the enzymatic mechanism of thioester hydrolysis and the structural basis for the different substrate specificities of the thioesterases. Clues to the mode of interaction between thioesterase II and the fatty acid synthase may also be revealed by a detailed structural analysis.